A Highly Multiplexed, Multiomic 3D Mouse Brain Map Using MALDI-IHC

使用 MALDI-IHC 绘制高度多重、多组学 3D 小鼠脑图

• 批准号: 10603396
• 负责人: Mark Lim
• 金额: $ 108.41万
• 依托单位: AMBERGEN, INC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10603396
• 关键词: 3-Dimensional; Agar; Antibodies; BRAIN initiative; Biological Markers; Brain; Brain imaging; Collaborations; Computer software; Data Analyses; Data Set; Development; Disease; Fluorescence; Freezing; Goals; Hospitals; Hour; Human; Image; Image Analysis; Imaging Device; Imaging technology; Immunohistochemistry; Individual; Label; Lectin; Length; Link; Maps; Metals; Methods; Modality; Molecular; Mus; Neurodegenerative Disorders; Neurosciences; Pattern; Phase; Play; Polysaccharides; Procedures; Production; Proteins; Proteomics; Resolution; Scanning; Slice; Spatial Distribution; Specimen; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stains; Techniques; Technology; Tissue imaging; Tissues; United States National Institutes of Health; Validation; Visualization; Woman; base; brain dysfunction; brain tissue; commercialization; denoising; fluorescence imaging; glycosylation; instrumentation; mass spectrometric imaging; medical schools; metabolomics; multimodality; multiple omics; neuronal circuitry; novel; preservation; protein expression; reconstruction; small molecule; tool

Summary/Abstract A central goal of the NIH Brain Initiative is to develop new imaging tools sufficiently powerful to spatially map at high resolution the neuronal circuitry and underlying molecular composition of the brain. While cutting edge imaging tools and related labeling techniques have been developed, it is still a major challenge to map the spatial distribution at different length scales of the thousands of biomolecules, including expressed proteins, which play key roles in brain function. The goal of this Phase II project is to evaluate the ability of a new tissue imaging technology developed by AmberGen, termed MALDI-IHC, to rapidly create a highly multiplexed, multiomic and multimodal 3D molecular map of the mouse brain. The development of MALDI-IHC for whole brain imaging will provide neuroscientists with an important new tool for exploring the underlying molecular basis of brain function and neurodegenerative disorders. MALDI-IHC is based on the use of novel photocleavable mass-tags (PC-MTs) developed by AmberGen which when linked to antibody or lectin probes enable targeted biomolecules to be identified in the mass spectrometric image. This approach significantly exceeds the multiplex capability of fluorescence immunohistochemistry (IHC) and previous cleavable mass-tag based methods which are generally limited to 5 biomarkers or require extensive cycling procedures. It also exceeds the capability of metal-tagged antibody techniques such as IMC and MIBI which can probe small mm2 regions at subcellular resolution but are limited to approximately 40 antibody probes and require several days to scan a whole tissue section. In contrast, MALDI-IHC can image an entire mouse brain FFPE section for over 100 targeted proteins at 40 µm resolution in less than one hour. The ability of MALDI-IHC to perform label-free, untargeted small molecule mass spectrometric imaging (MSI), fluorescence imaging using unique dual-labeled fluorescent-PC-MT probes and high-plex imaging of intact expressed proteins including glycosylation patterns on the same tissue section greatly extends the power of this approach. During Phase I, we demonstrated the feasibility of this combined approach on mouse brain FF and FFPE tissue specimens. During Phase II, we will develop methods using MALDI-IHC to reconstruct whole mouse brain protein expression maps at 40 µm voxel resolution. FFPE sagittal and coronal mouse brain tissue slices from mouse brain will be probed by MALDI-IHC using a panel of 50 NeuroMab PC-MT antibodies and 25 PC-MT lectins. Validation of individual PC-MT probes will be performed by comparing MALDI-IHC and fluorescence IHC images. A 3D tri-modal map of the mouse brain merging both metabolites and expressed proteins will also be reconstructed based on a demonstrated workflow that involves MSI of unlabeled small molecules from successive FF specimens, IHC staining with a 75-plex panel of PC-MT probes including some dual-labeled PC-MT antibodies, and fluorescence imaging followed by MSI of the PC-MTs. Reconstruction of 3D maps, visualization and image analysis will be performed using Bruker SCiLS™ software. Commercialization of MALDI-IHC technology will be accelerated by a close collaboration with Bruker Daltonics, the market leader of MALDI-MSI instrumentation.

摘要/摘要 NIH大脑倡议的一个中心目标是开发新的成像工具，这些工具足够强大，可以在 高分辨率大脑的神经回路和潜在的分子组成。在尖端的同时 成像工具和相关的标记技术已经发展起来，但绘制空间地图仍然是一个重大挑战 数以千计的生物分子在不同长度尺度上的分布，包括表达的蛋白质，它们发挥着 在大脑功能中的关键作用。这个第二阶段项目的目标是评估一种新的组织成像的能力 由AmberGen开发的技术，称为MALDI-IHC，用于快速创建高度多元化的、多组的和 小鼠大脑的多模式3D分子图谱。全脑成像用MALDI-IHC的发展将 为神经科学家探索大脑功能的潜在分子基础提供了一个重要的新工具 和神经退行性疾病。MALDI-IHC是基于新型可光裂解质量标签(PC-MTS)的使用 由AmberGen开发，当连接到抗体或凝集素探针时，使目标生物分子能够 在质谱学图像中被鉴定。这种方法大大超过了 荧光免疫组织化学(IHC)和以前的基于可切割质量标记的方法通常是 限制为5个生物标志物或需要广泛的自行车程序。它也超过了金属标签的能力 抗体技术，如IMC和MIBI，可以亚细胞分辨率探测小的mm2区域，但 限于大约40个抗体探针，需要几天时间才能扫描整个组织切片。相比之下， MALDI-IHC可以在40微米的分辨率下对超过100个目标蛋白质的整个小鼠脑FFPE切片进行成像 不到一个小时。MALDI-IHC进行无标记、非靶向小分子质谱学的能力 成像(MSI)、使用独特的双标记荧光PC-MT探针的荧光成像和高复合成像 完整表达的蛋白质，包括糖基化模式，在同一组织切片上大大扩展了力量 这种方法的一部分。在第一阶段中，我们在小鼠脑FF上证明了这种联合方法的可行性。 和FFPE组织标本。在第二阶段，我们将开发使用MALDI-IHC重建整体的方法 40微米体素分辨率的小鼠脑蛋白质表达图谱。FFPE小鼠矢状面和冠状面脑组织切片 将由MALDI-IHC使用一组50个NeuroMab PC-MT抗体和25个PC-MT来探测来自小鼠大脑的 凝集素。将通过比较MALDI-IHC和荧光IHC对单个PC-MT探针进行验证 图像。小鼠大脑的3D三模式图融合了代谢物和表达的蛋白质，也将是 基于涉及来自连续的未标记小分子的MSI的演示工作流重建 FF标本，用包括一些双标记PC-MT抗体的PC-MT探针的75个复合体进行IHC染色， 以及PC-MTS的荧光成像和MSI。三维地图、可视化和图像的重建 分析将使用Bruker SCiLS™软件进行。MALDI-IHC技术的商业化将是 通过与MALDI-MSI仪器的市场领先者Bruker Daltonics的密切合作，加速了这一进程。